Targeting Helicobacter pylori urease activity and maturation: In-cell high-throughput approach for drug discovery

Background: Helicobacter pylori is a bacterium strongly associated with gastric cancer. It thrives in the acidic environment of the gastric niche of large portions of the human population using a unique adaptive mechanism that involves the catalytic activity of the nickel-dependent enzyme urease. Targeting urease represents a key strategy for drug design and H. pylori eradication. Method: Here, we describe a novel method to screen, directly in the cellular environment, urease inhibitors. A ureolytic Escherichia colt strain was engineered by cloning the entire urease operon in an expression plasmid and used to test in-cell urease inhibition with a high-throughput colorimetric assay. A two-plasmid system was further developed to evaluate the ability of small peptides to block the protein interactions that lead to urease maturation. Results: The developed assay is a robust cellular model to test, directly in the cell environment, urease inhibitors. The efficacy of a co-expressed peptide to affect the interaction between UreF and UreD, two accessory proteins necessary for urease activation, was observed. This event involves a process that occurs through folding upon binding, pointing to the importance of intrinsically disordered hot spots in protein interfaces. Conclusions: The developed system allows the concomitant screening of a large number of drug candidates that interfere with the urease activity both at the level of the enzyme catalysis and maturation. General significance: As inhibition of urease has the potential of being a global antibacterial strategy for a large number of infections, this work paves the way for the development of new candidates for antibacterial drugs.